The major hypothesis guiding this grant has been that regulated synthesis of specific phosphoinositides at distinct cellular locations plays a critical role in cellular regulation. When this grant was initiated more than 10 years ago only two phosphorylated forms of phosphatidylinositol were known to exist in eukaryotic cells, PtdIns-4-P and PtdIns-4,5-P2 and these lipids were thought to be used exclusively for the generation of the second messengers Inositol-1,4,5-trisphosphate and Diacylglycerol. Largely through work supported by this grant, the total number of phosphoinositides known to exist in mammalian cells has expanded from two to seven and the number of families of phosphoinositide kinases has likewise expanded. It is now clear that many signaling proteins have evolved domains that directly bind to and discriminate between phosphoinositides as a mechanism of regulated recruitment to membranes. During the next funding period we will investigate how enzymes involved in PtdIns-4-P and PtdIns-4,5-P2 synthesis are located at specific subcellular sites and regulated by growth factors and hormones. However, we will place a major focus on a our surprising discovery of a new PtdIns-5-P signal transduction pathway in mammalian cells. The proposal has three specific aims. 1) We will determine how the PtdIns-5-P pathway is regulated by growth factors and other cellular regulators and determine its role in PtdIns-4,5-P2 production. We will explore the possibility that PtdIns-5-P is itself a second messenger. The gene encoding the PtdIns-5-P 4-kinase will be deleted in the mouse to determine the importance of this pathway at both the cell and animal level. 2) We will continue our efforts to define the types of PtdIns 4- kinases and focus on obtaining a cDNA clone of the major plasma membrane PtdIns 4-kinase of mammalian cells. 3) We will further investigate the regulation of Type I PtdIns-4-P 5-kinases and investigate their role in regulating actin remodeling at specific cellular locations.